The long range goal of this project is to understand the mechanisms by which the chromosome and low copy plasmids P1 and F of E. coli segregate into daughter cells. Dr. Wright proposes two approaches to study this process. In the first, he will isolate mutants with defects in the segregation process in order to identify genes and gene products which are involved. He has developed a direct selection for such mutants which makes use of cells carrying derivatives of the low copy plasmids P1 and F which carry genes that can be selected against. Selection for simultaneous loss of both plasmids yields segregation mutants with defects in either plasmid segregation or in plasmid and chromosome segregation. The method will be used to isolate a range of mutations which will be characterized by mapping, sequence analysis and by function of the deduced gene products. This approach should allow him to define many of the components of the segregation machinery. In the second approach, he will use a derivative of Green Fluorescent Protein, fused to the lac repressor (LacI) which binds to lac operator DNA to study chromosome and plasmid behavior in living cells. By placing an array of lac operator sequences at different sites on the chromosome, he will be able to examine sites that are replicated at different times and that may be localized differently in the cell. He will examine plasmids P1 and F, carrying lac operator sites, in the same way. The resolution of the technique should allow him to determine when particular sites on the chromosome are duplicated, when plasmids are duplicated, when they separate from one another and where they become localized within the cell. He will utilize the same technique to characterize mutants isolated by the first approach. This approach should yield new information about the components of the segregation apparatus in bacteria which must include elements that specify position within the cell. Such positional information is crucial for proper growth and division in all cells. The present approach presents an opportunity to identify positional elements in bacteria, about which little is currently known.